System-wide biochemical analysis reveals ozonide antimalarials initially act by disrupting Plasmodium falciparum haemoglobin digestion
Fig 4
Peroxide antimalarials act by perturbing haemoglobin digestion.
a, Disruption of protease abundance in the haemoglobin digestion pathway [36]. Values are the average log2 fold change (± SD) relative to the untreated control of at least three biological replicates. Trophozoite infected cultures (P. falciparum 3D7) were incubated with OZ277, OZ439 (both 300 nM) or DHA (100 nM) for 3 h. Falcipain 2 (FP 2) was identified in only two OZ277 treatment experiments, therefore the mean alone is shown. Aminopeptidase P (PfAPP) was not identified in any of the proteomic experiments. DPAP1, dipeptidyl aminopeptidase 1; FP, falcipain; HAP, histo-aspartic protease; PfAPP, aminopeptidase P; PfA-M1, alanyl aminopeptidase; PfA-M17, leucyl aminopeptidase; PfM18APP, aspartyl aminopeptidase; PM, plasmepsin; * P-value < 0.05. b, Parasite cysteine protease activity after peroxide treatment using the activity-based probe (ABP), DCG04. Cysteine protease activity and densitometric analysis of the falcipain (FP) 2/3 and dipeptidyl aminopeptidase 1 (DPAP1) signal after OZ277 and OZ439 treatment in P. falciparum trophozoite stage parasites (3D7) using DCG04 in lysates at pH 5.5 (acidic). Trophozoite infected cultures were incubated with OZ277, OZ439 (both 300 nM) or an equivalent volume of DMSO (control). DCG04 labelling was detected by blotting membranes with streptavidin-AF647 after SDS-PAGE and transfer. The lanes for each time point are independent drug treatments and represent at least three biological replicates per time point that were run on the same gel side-by-side. For the densitometric analysis, the post drug treatment FP 2/3 and DPAP1 signal intensity was normalised to the average signal intensity of the appropriate time point in the untreated control (±SD). c, Schematic showing that infected RBCs treated with DHA or ozonides (OZ) can use exogenous amino acids when cultured in AA medium (Full RPMI medium with all 20 amino acids) in response to disrupted haemoglobin digestion (arrow shown in blue), while parasites in Iso medium (supplemented with isoleucine alone at a final concentration of 147.5 μM) must rely solely on haemoglobin digestion for amino acids. d, Amino acid requirement for cultured P. falciparum 3D7 parasites. Parasite viability measured following 48 h incubation in medium containing all amino acids (AA, black bars) and isoleucine alone medium (Iso, grey bars). e, Parasite sensitivity to peroxides when cultured in AA (black bars) medium compared to Iso (grey bars) medium. Trophozoite infected cultures (3D7) were incubated with pyrimethamine (PYR), OZ277, OZ439 (all 300 nM), DHA (100 nM) or an equivalent volume of DMSO (control) for 3 h. Data represents the mean ± SD of at least three biological replicates. Growth values for each treatment is expressed relative to the appropriate untreated medium (Iso or AA) control, which was set to 100%. * P-value < 0.05.